Changes in neuronal activity of the inferior colliculus in rat after temporal inactivation of the auditory cortex

The role of cortico-tectal pathways in auditory signal processing was studied in anesthetized rats by comparing the extracellular single unit activity in the inferior colliculus (IC) before and after functional ablation of the auditory cortex (AC) by tetrodotoxin (TTX). The responses of several IC neurons to sound stimuli were simultaneously recorded with a 16-channel electrode probe introduced into the IC. Click-evoked middle latency responses (MLR) recorded from the AC were suppressed for several hours after TTX injection. During AC inactivation the firing rate of IC neurons increased (40 % of neurons), decreased (44 %) or did not change (16 %) in comparison with control conditions. In several IC neurons, TTX injection resulted in alterations in the shape of the rate-level functions. Response thresholds, tuning properties and the type of discharge pattern of IC neurons were not altered during AC inactivation. However, in one-third of the neurons, the initial part of the response was less altered than the later, sustained part. In two-thirds of neuronal pairs, functional decortication resulted in a change in the cross-correlation coefficient. The results reveal the complex changes that appear in IC neuronal activity after functional ablation of the ipsilateral auditory cortex.     

Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis

Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.     

Treatment of synchronized K562 cells by tetrafluoroaluminate does not modulate the fluorescence of ethidium bromide and 4'6-diamidine-2-phenylindole in case of the binding with nucleoid DNA

Earlier we showed that 4-hours treatment of cells K562 with the GTP-binding protein activator AlF4- (10 mM NaF + 20 microM AlCl3) increased the DNA fragmentation on an average to 5% of the total 3H-thymidine-labeled DNA. The viability of cells under these conditions did not change. It has been suggested that gene toxic action of AlF4- is a result of cell proliferation block induced by AlF4-. In the present work we tried to determine possible changes in the ethidium bromide and 4',6-diamidine-2-phenylindole (DAPI) fluorescence when they bind with nucleoid DNA of synchronized cells K562 treated with AlF4-. Cells K562 were incubated for synchronization with 2 mM thymidine during 15 hours. Under these conditions DNA synthesis (3H-thymidine uptake) was suppressed by 94-99%. It has been found that the treatment of "cool" thymidine-incubated cells K562 with AlF4- did not change the fluorescence of either ethidium or DAPI. The presence of phorbol-12-myristate-13-acetate (PMA) in the incubation medium did not influence the results. On the other, hand the rat thymocytes incubated with dexametazone (2 microM) during 4 hours (positive control of DNA fragmentation) demonstrated the increase in both parameters. PMA decreased the ethidium fluorescence that correspond to its (PMA) ability to suppress fragmentation of thymocyte DNA. On the base of the results we suggested that AlF4- did not induce DNA fragmentation in the cells K562 with the blocked DNA synthesis.     

Effect of the antioxidant dibunol on the function of the adrenal cortex, the thyroid and the adenohypophysis in adult and old rats

The effect of a single antioxidant (dibunol-D) injection (100 mg/kg body weight) on the functional activity of adrenal cortex (AC), thyroid gland (TG) and tropic hormone production by adenohypophysis (AH) has been studied in old and adult rats. For 48 hours following D administration two-phase changes in adrenocorticotropic function of AH and steroidogenesis were detected in the AC: activation during the first hours was followed by suppression 24 hours later, and recovery 48 hours later. Thyrotropic AH function and thyroidogenesis were found to be decreased during the first hours of D effect. Thyroidogenesis recovery by the end of the first day was delayed as compared to the recovery of AH thyrotropic function. It is suggested that the mechanisms of D action are based on its effects mediated by changes in the functional activity of endocrine glands and associated with resetting of endocrine regulation of body functions.     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.     

Physicochemical stability of cimetidine amorphous forms estimated by isothermal microcalorimetry

The effect of humidity on the physicochemical properties of amorphous forms of cimetidine was investigated using differential scanning calorimetry, isothermal microcalorimetry, and x-ray diffraction analysis. Amorphous forms were obtained by the melting (amorphous form M [AM]) and the cotton candy (amorphous form C [AC]) methods. Thermal behaviors of AM and AC with or without seed crystals were measured using an isothermal microcalorimeter under various conditions of relative humidity (RH) and temperature, respectively. The crystallization kinetics of amorphous solids was analyzed based on 10 kinds of solid-state reaction models. AM transformed into form A at 11% RH, 50°C but transformed into a mixture of form A and monohydrate at 51% and 75% RH at 25°C. The mean crystallization times (MCTs) of the heat flow curve of AM and AC at 11% RH, 50°C were 47.82 and 32.00 hours, respectively, but at 11% RH, 25°C both were more than 4320 hours. In contrast, AC transformed into form A under all storage conditions. The MCTs of AC at 51% and 75% RH were 29.61 and 11.81 hours, respectively; whereas the MCTs of AM were 46.79 and 15.52 hours, respectively. The crystallization of amorphous solids followed the three-dimensional growth of nuclei (Avrami equation) with an induction period (IP). The IP for AM at 11% RH, 50°C was more than 2 times that for AC, but the difference in the crystal growth rate constant (CR) between AC and AM was within 10%. The IP for AM at 75% RH, 25°C was reduced to only 10% of the IP at 51% RH with increasing humidity, but the CR did not change significantly. In contrast, the IP for AC was slightly reduced at 75% RH compared with 51% RH, but the CR was about 5 times greater. At 75% RH, 25°C, the IP and CR of AM were about one-fourth the values of AC. This result suggests that the crystallization process consists of an initial stage during which the nuclei are formed and a final stage of growth.     

Effects of Y. pestis mouse toxin on carbohydrate metabolism in rats

Effects of intravenous Y. pestis mouse toxin (LD50) injection on glucose, lactate glucagon, insulin blood levels and cAMP liver content in dynamics of intoxication development were studied. Hypoglycemia, observed 2 hours after toxin administration seems not to be due to the enhanced glucose utilization in peripheral tissues because insulin blood level during this period was decreased and lactate concentration has not been changed. Glucagon content by 2-5 hour of shock was strong elevated. Proposal is made that Y. pestis mouse toxin might induce carbohydrate metabolism alterations via direct liver glucose synthesising enzymes inhibition rather than cAMP-dependent glycogenolysis and gluconeogenesis regulation disturbances in this organ.     

Comparative-physiological study of leukocyte participation in initiation of lipid peroxidation during nitrite intoxication in rats and muskrats

The study is carried out on Wistar white rats non-adapted to oxygen deficit and on semiaquatic rodents muskrats adapted to periodic arrest of respiration during diving under conditions of Nembutal narcosis. It has been revealed that 1 h after a subcutaneous injection of sodium nitrite (3 mg/100 g body mass), intensification of lipid peroxidation (LPO) in the muskrat brain is absent, the activity of the antioxidant enzyme catalase increasing 16 times (p < 0.01) as compared with control injected with equivalent saline volume. In heart and liver, there was a statistically significant decrease of the content of LPO products active in the test with 2-thiobarbituric acid; in the femoral muscle tissue, the LPO intensity did not change. In rats, unlike muskrats, after injection of sodium nitrite, an increase of LPO is recorded in brain, while a decrease of the LPO product content in the femoral muscle; in liver the LPO intensity did not change. In muskrats, the sodium nitrite administration led to a decrease of the leukocyte spontaneous mobility, of lymphocyte cytokine-producing activity, and of neutrophil bactericidal activity (by the content of cationic proteins in neutrophilic phagocytes), whereas in rats the leukocyte mobility did not change, only the blood neutrophil bactericidal activity decreased. The ability of neutrophils to produce the superoxide anion during the nitrite intoxication did not change both in rats and in muskrats. The obtained data allow concluding that under conditions of Nembutal narcosis the leukocyte functional activity on the background of nitrite intoxication is suppressed to the greater degree in the muskrats genotypically adapted to oxygen deficit than in immunocompetent cells of the rodents not adapted to hypoxia.     

Acute renal insufficiency in the rabbit by glycerol.

The appearance of an acute renal insufficiency in the rabbit, after glycerol injection (10, 13 or 15 ml/kg of a 50% solution) is investigated. After a 24 hours of intoxication, especially in the ten following days, cylinders, erythrocytes and renal cells appear in the urine sediment. Proteinuria appears after 24 hours and practically disappears after 72 h. Glucosuria persists from 24 hours to 6 days. Haemoglobinuria is intense after 24 and 48 hours and persists slightly about 6 days. Na, K and Cl elimination in urine diminishes clearly in all animals. Plasma K increases in non-surviving animals and does not change in those surviving. Plasma Na does not change in the dying ones, and decreases in those surviving. In non-surviving animals, pH, pCO2 and CO3H minus decrease sharply. In the surviving ones pCO2 decreases clearly after 24 hours, increasing afterwards slowly to normal values. pH increases, slightly during the first 48 hours, and then neatly during approximately 6 days. Standard CO2H minus does not change during the first 48 hours, increasing afterwards during 6 to 7 days. Histologically, the chief lesion is a vacuolar degeneration of the proximal tubule. The possible mechanisms of such alterations are discussed.     

Alpha tocopheryl succinate inhibits melanocyte-stimulating hormone (MSH)-sensitive adenylate cyclase activity in melanoma cells

D-alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B-16 melanoma cells, reduced basal and melanocyte-stimulating hormone (MSH)-stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride- and forskoline-stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 micrograms/ml] for a period of 24 hours was sufficient to reduce MSH-stimulated AC activity. Other forms of vitamin E, such as d1-alpha tocopheryl nicotinate, d1-alpha tocopheryl acetate, and d1-alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH-stimulated AC activity. Retinoic acid, which inhibited the growth of B-16 melanoma cells, also reduced basal and MSH-, NaF-, and forskolin-stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH-stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH-sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.     

Effects of antrodia camphorata on viability, apoptosis, and [Ca2+]i in PC3 human prostate cancer cells

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.     

The effect of delta sleep-inducing peptide on the convulsive activity in corasol kindling

The influence of intraperitoneal delta-sleep inducing peptide (DSIP) injection (100 micrograms/kg) on the epileptic activity was investigated in the experiments on Wistar rats and (CBA X C57B1/6)F1 mice. The model of chronically developing epileptic activity--the model of pharmacological kindling--was created by daily repeated corasole injections in subconvulsive doses (30 mg/kg). It has been shown that DSIP injection delayed the manifestation of generalized seizures during kindling, led to the suppression of seizure activity and reduced the mortality rate of animals that developed kindled seizures. The antiepileptic effect of DSIP was observed throughout the period of 5 minutes to 24 hours after the injection. Naloxone (2.5 mg/kg) did not change the antiepileptic effect of DSIP.     

Internalization of the cytotoxic molecules of T101 F(ab')2-(ricin-A-chain) immunotoxin into human T-leukemic cells

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab')2-T101(ricin-A-chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells. We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy-dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate-limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low. Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.     

Prostaglandins and cyclic nucleotides in the dynamic development of an experimental poisoning syndrome caused by Yersinia pestis lipopolysaccharide

This work deals with the influence of Y. pestis lipopolysaccharide (LPS), introduced intraperitoneally in a dose of 2 LD50, on the content of prostaglandins (PG), such as PGE, PGF2 alpha and 6-keto-PGF1 alpha, thromboxane, cAMP and cGMP in the liver, lungs and blood plasma of guinea pigs in the process of the development of experimental intoxication. The content of thromboxane in blood plasma increased 2.4-fold in 2 hours after intoxication and remained elevated for as long as 5 hours. Other parameters of blood plasma remained unchanged. The data obtained in this investigation indicate that thromboxane, known as a regulator of thrombogenesis, may induce early disturbances in microcirculation. A change in the content of PG was shown to occur in pulmonary tissue 2 and 5 hours after the beginning of intoxication. The content of PG in liver tissue was found to occur at a later period of the toxic action. The concentration of cyclic nucleotides (CN) in the tissues under study sharply increased even at the initial stage of the development of shock in guinea pigs. The effect of LPS on the metabolism of PG and CN, revealed in this investigation, resembles the effect produced by the thermostable fraction of "mouse" toxin.     

Protective reaction of the myocardium in poisoning

Intensive synthesis of collagen-like substance was revealed in the rabbit myocardium during experimental diphtheria intoxication. It was more marked in the right ventricle 24 hours after the injection of diphtheria toxin. Since similar changes (the substance was mainly formed around blood vessels) have been observed in other cases of toxic myocardial alterations (i.e. ethanol intoxication, injection of pharmacological agents, etc.), it can be assumed that it is a standard protective reaction of the altered heart to the penetration of toxic agents from the blood into the myocardial tissue.     

Type IV collagen reduces mucin 5AC secretion in three-dimensional cultured human primary airway epithelial cells

Mucin 5AC (MUC5AC) hypersecretion induces airway narrowing in patients with asthma, which leads to breathing problems. We investigated the regulation of MUC5AC secretion by extracellular matrix (ECM) proteins in human primary airway epithelial cells from patients with asthma. The addition of type IV collagen to three-dimensional cultured human primary airway epithelial cells, which mimics the airway surface, reduced MUC5AC secretion in the medium, while the addition of laminin increased MUC5AC secretion. Furthermore, the addition of fibronectin did not affect MUC5AC secretion. In particular, the repeated addition of a low concentration of type IV collagen demonstrated a cumulative effect on the reduction in MUC5AC secretion. Human primary cells incubated with type IV collagen showed downregulated extracellular signal-regulated kinase (ERK) activity, which induced MUC5AC hypersecretion but did not affect Akt activity. These results suggest that the addition of type IV collagen to the apical surface of primary cells downregulates MUC5AC secretion and has a cumulative effect on MUC5AC secretion which might be effected via the ERK signaling pathway.     

Changes in Na+, K+-adenosinetriphosphatase and 5'-nucleotidase activities in cell membrane isolated from rat liver after acute white phosphorus poisoning

Na+, K+-ATPase and 5'-Nucleotidase activities in rat liver plasmamembranes after "in vivo" intoxication with a single dose of white phosphorus (10 mg/kg b.w. "per os") are investigated. Na+, K+-ATPase activity is significantly increased 1 hour and inhibited 12 hours after intoxication. 5'-Nucleotidase is strongly increased at 1, 2 and 4 hours after poisoning and is significantly decreased at 12 hours. The enhancement of both the enzymatic activities is evident prior to triglyceride accumulation in rat liver. Our results suggest that lipid fluidity of cell membrane is early and mildly affected during white phosphorus poisoning.     

Comparative fluorometric study of benzo(a)pyrene and aza-aromatic hydrocarbon penetration and metabolism kinetics in the skin of hairless mice.

The kinetics of penetration and metabolism of BaP, BACs, DB(a,h)ACs and DB(c,g)Cs in the skin of hairless mice was studied. The relative fluorescence intensities were measured during three hours after applying 10 nmoles of the compound to the interscapular region of the mice. By using a kinetical model which combines a non-steady diffusion of a hydrocarbon through the stratum corneum and the metabolic oxidation by epidermal cells, the rate constants for the two processes were calculated. It has been shown that B(a)AC, B(c)AC and 12-MB(a)AC penetrate into the skin and are oxidized by epidermal cells more efficiently than BaP. In contrast, alkyl-DB(a,h)ACs (except 14-MDB(a,h)AC) show a great stability in the mouse skin. The carcinogenic BaP, 7-MB(c)AC and DB(a,h)AC have average rates of elimination from the skin.     

Acute toxicity and hematological and serum changes caused by various cobalt salts in rats

The acute toxicities of chloride, acetate, cobalt nitrate and cobalt sulphate (II) were investigated in rats. The values obtained for the LD50 (7 days) were 133, 194, 198 and 279 mg of Co/kg respectively, when the salts were given orally. The values for intraperitoneal administration were 10.6, 8.8, 8.3 and 11.5 mg of Co/kg. The majority of deaths occurred during the first 48 hours. The physical and clinical signs appearing after the intoxication disappeared for the most part after the first 72 hours. Also, the effect produced by chloride and cobalt acetate (II) given orally and intraperitoneally, in some hematological parameters and serum parameters during the first 24 hours after intoxications, has been studied at several doses. A noteworthy increase was observed in the hemoglobin and hematocrit as well as in the plasma proteins. There was significant hyperglycemia and also significant changes in the lipid parameters such as triglycerides and cholesterol. The duration and degree of the change depended on the dose. As a general rule, no significant differences were registered between the results obtained for the two salts.